Aspartate is an important excitatory transmitter in the brain, as well as a protein component and metabolic intermediate. However, few experimental approaches exist by which the transmitter and metabolic roles can be separated for examination. Using lesion studies, it was shown that aspartate is involved in the perforant pathway to the hippocampus. Since only a few areas of brain have a dense enough innervation by aspartergic neurons for the lesion method to be practical, an aspartate binding method was developed for use in all brain areas. Two binding sites were demonstrated, differing in their sodium dependence, Kd, inhibition by other amino acids, and in their regional distribution. The ability to measure neuronal binding sites (receptors) opens up the possibility of studying interactions with other neurotransmitters, cotransmitters and neuromodulators, as well as the possibility of exploring aspartate pathways in areas of brain where detection of changes in aspartate content would not be possible.